The long term goal of this project is to understand, at a molecular level, how adenylate cyclase is regulated in the yeast, S. cerevisiae. Intracellular levels of cAMP have been shown to play a major role in the regulation of growth in yeast. Production of cAMP is catalyses by adenylate cyclase system is thus relevant not only to mammalian adenylate cyclase, which plays a crucial role in many homeostatic mechanisms, but to the proteins encoded by the animal ras oncogenes as well. We propose to identify the proteins that make up the enzyme complex, assign functions to these proteins, and determine the locations of functional domains on the catalytic protein. In order to identify proteins associated with adenylate cyclase, we will rely on biochemical techniques, including coprecipitation using antibodies to adenylate cyclase, crosslinking with bifunctional agents, and affinity purification using segments of the catalytic subunit overexpressed in E. coli. We will examine the functional roles of these proteins by using molecular genetic techniques to produce yeast strains which lack each protein, but which retain the other components of the system. We will determine how these changes in the subunit composition of adenylate cyclase affect the biochemical properties of the enzyme, as well as the phenotypes of the altered yeast. In order to produce the altered strains, we will clone the genes for proteins associated with adenylate cyclase, and use the cloned DNA to disrupt the chromosomal copy of each gene in wild type yeast. To examine the system in more detailed, we will identify the regions of the catalytic protein that are important in interactions with the RAS proteins, the cell membrane, and other proteins. Specific, in frame deletions will be made in the coding region of the CYR1 gene, encoding the catalytic polypeptide, and the resulting truncated enzyme will be examined for losses in function. An alternate strategy will be to express isolated segments of the catalytic protein at high levels in wild type yeast. If these segments fold properly to produce functional domains, they will compete with the homologous domains on the normal enzyme, and prevent interactions with proteins that normally associate with adenylate cyclase. This will provide positive evidence for the locations of functional domains within the catalytic polypeptide.